Application of Cslnc924 gene in diagnosis or prevention and treatment of tea tree anthracnose

Abstract

The invention discloses application of a Cslnc924 gene in diagnosis or prevention and treatment of tea tree anthracnose, and relates to the technical field of prevention and treatment of tea tree anthracnose. The Cslnc924 gene is used for promoting the expression of the Cslnc924 gene so as to enhance the resistance of tea trees to colletotrichum gloeosporioides, and the cDNA full-length sequence of the Cslnc924 gene is as shown in SEQ ID No.1. The method has the beneficial effects that on the basis of an agrobacterium tumefaciens-mediated transient overexpression technology and an antisense oligonucleotide-mediated gene silencing technology, the Cslnc924 gene is overexpressed and silenced on tea tree leaves respectively, and an anthrax inoculation test is carried out. Results show that when Cslnc924 is overexpressed, compared with a control group, CsmiR390 is remarkably up-regulated, downstream CsARFs (CsARF2.1, CsARF2.2, CsARF3 and CsARF4.1) of CsmiR390 are remarkably down-regulated, at the moment, resistance of tea trees to colletotrichum gloeosporioides is enhanced, and the area of disease spots is remarkably reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of tea tree anthracnose prevention and treatment, and particularly to an application of a Cslnc924 gene in diagnosing or preventing tea tree anthracnose. Background Art

[0002] Anthracnose is a serious semi-lethal fungal disease of tea leaves that causes great harm to tea production.

[0003] Increasing evidence supports the key role of non-coding RNA genes in enhancing plant immunity. The mechanism of action of lncRNA in resistance regulation is gradually being elucidated. For example, the long non-coding RNA (lncRNA) ALEX1 in rice directly binds to the intrinsically disordered middle region of ARF3 to promote ARF3 homodimerization, thereby enhancing its ability to transcriptionally repress the expression of downstream target genes (such as JAZ13), regulating the jasmonic acid signaling pathway and enhancing pathogen resistance in rice. As an important ncRNA, miRNA also plays an indispensable role in plant defense mechanisms. An example is that the miRNA Os-miR169y induced by Sclerotinia sclerotiorum in rice can target the 60S ribosomal protein L19 (SsRPL19) of Sclerotinia sclerotiorum, affecting the ribosome assembly of the pathogen, thereby inhibiting the growth and pathogenicity of the pathogen. NB-LRR (nucleotide binding and leucine rich repeat) genes are the main type of plant innate immune receptors. A large number of miRNAs have been found to regulate the expression of NB-LRR genes in different plants. These miRNAs trigger phasi-RNA synthesis by targeting NB-LRR genes to enhance their silencing effect. The participation of mRNA and ncRNA interactions is indispensable in conferring resistance traits on tea plants, but research on their mechanism of action is still very limited. We previously identified an evolutionarily conserved lncRNA OPRL in tea plants, which negatively regulates tea plants' resistance to anthracnose by forming a triplex with the target gene OPR to inhibit JA synthesis.

[0004] For example, lncRNA 81246 can act as a ceRNA to influence the miR164d-CsNAC1 regulatory module to negatively regulate tea plant resistance to tea leaf spot. We performed sRNA transcriptome sequencing on tea leaves infected with the anthracnose fungus C. camelliae and analyzed the differentially expressed miRNAs and their target genes using GO and KEGG pathway enrichment analysis, confirming that plant-pathogen interaction and plant hormone signal transduction pathways are regulated by miRNA-target networks.

[0005] Defects and shortcomings of existing technology:

[0006] ① The existing tea tree anthracnose prevention technology relies on chemical control. The long-term and large-scale use of chemical agents such as pyraclostrobin emulsifiable concentrate, difenoconazole water-dispersible granules, and thiophanate-methyl wettable powder can easily lead to pesticide residues in tea leaves and soil, which not only affects the quality of tea, but also pollutes the ecological environment and threatens the balance of the tea garden ecosystem.

[0007] ② The research on the mechanism of LncRNA regulating anthrax resistance at the molecular level is not in-depth enough, and it is impossible to effectively prevent anthrax at the biomolecular level.

[0008] Chinese patent application document with publication number CN108718838A discloses a method for preventing and controlling tea anthracnose, including the following steps: (1) strengthening tea garden management, digging ditches and draining waterlogged tea gardens, and removing fallen leaves in autumn and winter; (2) appropriately increasing the application of phosphorus and potassium fertilizers, applying 30-35 kg of farmyard manure, 0.9-1.1 kg of urea, 0.5-0.7 kg of phosphorus fertilizer, 0.6-0.9 kg of nitrogen fertilizer, and 2-3 kg of potassium fertilizer per plant per year; (3) enhancing disease resistance and selecting disease-resistant varieties; (4) removing diseased leaves: promptly removing diseased branches and leaves; (5) drug control: in the early stage of the disease, evenly spraying 1000-1200 times of gray frost and Bacillus can be used, and tea leaves can be picked after 14-16 days. This patent can effectively prevent and control tea anthracnose, allowing tea trees to grow normally and ensuring tea quality and yield. However, this patent still remains at the level of physical or chemical drugs to prevent and control tea tree anthracnose, rather than the genetic level. It is time-consuming and labor-intensive, and has poor prevention and control efficiency, so it needs further improvement. Summary of the Invention

[0009] The technical problem to be solved by the present invention is to propose a new use of a gene in preventing and treating tea tree anthracnose.

[0010] The present invention solves the above technical problems through the following technical means:

[0011] In a first aspect of the present invention, a use of the Cslnc924 gene is proposed to promote the expression of the Cslnc924 gene to enhance the resistance of tea plants to anthracnose. The full-length cDNA sequence of the Cslnc924 gene is shown in SEQ ID No. 1.

[0012] Preferably, the Cslnc924 gene is a long non-coding RNA, and its RNA sequence is shown as SEQ ID No. 2.

[0013] The second aspect of the present invention proposes the use of a reagent for detecting the expression level of the Cslnc924 gene in diagnosing the strength of tea tree anthracnose resistance.

[0014] The third aspect of the present invention proposes the use of the Cslnc924 gene in the preparation of a drug for preventing and treating tea tree anthracnose.

[0015] A fourth aspect of the present invention proposes the use of the Cslnc924 gene in cultivating anthracnose-resistant tea varieties.

[0016] A fifth aspect of the present invention provides a drug for preventing and treating tea tree anthracnose, comprising an agent that promotes the expression of the Cslnc924 gene.

[0017] A sixth aspect of the present invention provides an anthrax-sensitive tea tree model, wherein the anthrax-sensitive tea tree model contains a product that inhibits the expression of the Cslnc924 gene.

[0018] The seventh aspect of the present invention provides an expression vector containing the above-mentioned Cslnc924 gene.

[0019] The eighth aspect of the present invention provides a method for breeding anthracnose-resistant tea trees, comprising the following steps: obtaining tea trees having higher anthracnose resistance than the target plant by promoting the expression of the Cslnc924 gene in the target plant.

[0020] Preferably, the method for promoting the expression of the Cslnc924 gene in the target plant comprises: constructing a Cslnc924 gene overexpression vector and introducing the vector into the target plant.

[0021] The beneficial effects of the present invention are:

[0022] 1. The present invention proposes the application of Cslnc924 gene in diagnosing or preventing anthracnose of tea trees. The 257bp-511bp of Cslnc924 gene binds to (-825bp)-(-1053bp) in the CsmiR390a promoter and activates the transcription of CsmiR390a ( Figure 1), positively regulating the miR390-TAS3-ARF2s module, reducing the expression of ARF2s (ARF2.1 and ARF2.2), thereby enhancing tea plant resistance to anthracnose. This study proposes a new use for the Cslnc924 gene and expands our understanding of the regulation of the miR390-TAS-ARF2s module.

[0023] 2. The present invention verifies whether Cslnc924 affects the resistance of tea plants to anthracnose by regulating the MiR390-TAS3-ARF2s module. Based on Agrobacterium-mediated transient overexpression technology and antisense oligonucleotide-mediated gene silencing technology, the Cslnc924 gene was overexpressed and silenced in tea leaves, respectively, and anthracnose inoculation experiments were carried out. The results showed that when Cslnc924 was overexpressed, compared with the control, the Cslnc924 and CsmiR390 genes were significantly upregulated, while their downstream CsARFs (CsARF2.1, CsARF2.2, CsARF3 and CsARF4.1) were significantly downregulated. At this time, the resistance of tea plants to anthracnose was enhanced, and the area of ​​lesions was significantly reduced ( Figure 2 ).

[0024] 3. In contrast, when Cslnc924 was silenced, the Cslnc924 and CsmiR390 genes were significantly downregulated, while the transcription levels of their downstream CsARFs (CsARF2.1, CsARF2.2, CsARF3 and CsARF4.1) were significantly upregulated. At this time, the resistance of tea plants to anthracnose was weakened and the lesion area was significantly increased. It is worth noting that when Cslnc924tu without the CsmiR390 promoter binding site was overexpressed in tea plants, the expression levels of CsmiR390a and its downstream CsARFs (CsARF2.1, CsARF2.2, CsARF3 and CsARF4.1) did not change significantly, and the lesion size was not significantly different from that of the control ( Figure 2 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1Figure 1 shows the experimental verification of Cslnc924 activating CsmiR390a in Example 1 of the present invention, where A is a schematic diagram of the gene structure of Cslnc924, CsmiR390a precursor, and CsmiR390a promoter. The red box indicates the reverse complementary sequence. B is a diagram of the molecular interaction model between Cslnc924 and the CsmiR390a promoter predicted by AlphaFold3 software. C is a diagram of the secondary structure of Cslnc924 and its truncated variant (Cslnc924tu), with the red box indicating the stem-loop structure of the complementary region. D is a schematic diagram of the truncated CsmiR390a promoter construct in a dual-luciferase reporter assay. E is a diagram showing the activation effect of Cslnc924 and Cslnc924tu on the truncated CsmiR390a promoter fragment in a dual-luciferase reporter assay.

[0026] Figure 2 Figures demonstrating the functional validation of Cslnc924 and Cslnc924tu in regulating tea plant disease resistance in Example 1 of the present invention. Figures A and B illustrate the functional analysis of Cslnc924 in tea plant resistance to anthracnose via transient Agrobacterium overexpression and AsODN-mediated gene silencing. Figure C demonstrates the regulatory effect of Cslnc924tu on tea plant resistance via transient Agrobacterium overexpression in tea leaves. qRT-PCR quantitative analysis of gene expression levels of CsmiR390a and CsARFs. Data are mean ± SD (n ≥ 6). Significance was assessed by two-tailed Student's t-test (*P < 0.05, **P < 0.01). Individual data points represent biological replicates. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below have the same meaning as those understood by professional and technical personnel in this field.

[0028] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources or prepared by known methods.

[0029] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples were repeated three times and the results were averaged.

[0030] Example 1:

[0031] This example provides a method for enhancing anthrax resistance by regulating the miR390 / TAS3 / ARFs module in tea plants using Cslnc924. The method comprises the following steps:

[0032] To verify whether Cslnc924 affects tea plant resistance to anthrax by regulating the miR390-TAS3-ARF2s module, we first confirmed through dual-luciferase reporter assays that Cslnc924 can bind to the CsmiR390a promoter through base complementation and activate its promoter activity.

[0033] The dual-luciferase reporter assay includes the following steps:

[0034] 1. Construction of overexpression vectors for Cslnc924 and its mutant forms

[0035] (1) RNA obtained from Zhongcha 108 was used as the material to synthesize 5′ and 3′ RACE-Ready cDNA templates required for RACE cloning using the SMARTer RACE 5′ / 3′ Kit (Takara). PCR amplification was performed using the following primers, referring to the instructions of the kit:

[0036] Cslnc924-5'RACE-R (SEQ ID NO:3): 3'-GGACATCTGCTCAAGATCACTGG-5';

[0037] Cslnc924-3'RACE-F (SEQ ID NO:4): 5'-GCTATAAGCTGCATTGTTAAGC-3';

[0038] Cslnc924-F (SEQ ID NO:5): 5'-AAGCAGTGGTATCAACGCAGAGTACA-3';

[0039] Cslnc924-R (SEQ ID NO:6): 3'-GCAGTGGTATCAACGCAGAGTAC-5';

[0040] The PCR reaction system was based on PrimeSTAR TM HSDNA Polymerase (Taraka) was used according to the instructions. The PCR amplification program was as follows: 98°C for 10 seconds, 62°C for 10 seconds, 72°C for 1 minute, 30 cycles, 72°C for 6 minutes, and 16°C for termination. The resulting PCR products were verified by agarose gel electrophoresis, and the bands at the target positions were recovered from the gel.

[0041] (2) Using the amplified product recovered in step (1) as a template, the following primers were used to perform base mutation on Cslnc924 to obtain the cloned product of Cslnc924tu:

[0042] Cslnc924tu-F1 (SEQ ID NO:7): 5'-ACATGGGCTACAACGGTCCGAC-3'

[0043] Cslnc924tu-R1 (SEQ ID NO:8): 3'-GGTGTTTTATTACCCACAGC-5'

[0044] Cslnc924tu-F2 (SEQ ID NO:9): 5'-GCTGTGGGTAATAAAACACC-3'

[0045] Cslnc924tu-R2 (SEQ ID NO:10): 3'-GCAGTGGTATCAACGCAGAGTAC-5'

[0046] (3) Take the 1305 vector and perform double digestion with restriction endonucleases BamhI and XbaI to recover the vector backbone;

[0047] (4) The amplified products recovered in steps (1) and (2) were connected to the vector backbone recovered in step (3) to obtain recombinant plasmids 35S::Cslnc924 and 35S::Cslnc924tu. The connection process was based on the ClonExpress II One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd. 2 μL of the recovered product was connected to 0.5 μL of the cloning vector, reacted at 25°C for 15 minutes, ice-bathed for 5 minutes, added 20 μL of DH5α competent medium, heat-shocked at 42°C for 1 minute, ice-bathed for 2 minutes, and the resulting product was added to 200 μL of LB medium and incubated in a 37°C shaker for 1 hour. Finally, the bacterial solution was spread on a plate containing Kan antibiotics and then cultured in a 37°C incubator. The colony growth was observed the next day, and a single colony was picked for PCR verification. The bacteria with the correct band position were sequenced.

[0048] 2. Construction of overexpression vectors of CsmiR390a promoter and its truncated forms

[0049] (1) Using the DNA obtained from Zhongcha 108 as the material, PCR amplification was performed using the following primers as described in step 1, and the amplified product was recovered:

[0050] ProCsmiR390a-F (SEQ ID NO:11):

[0051] 5'-GAGTGTGAGGTAACGTAGTCGGCAG-3'

[0052] ProCsmiR390a-R (SEQ ID NO:12):

[0053] 3'-TGAGTTTGTATATGTGATGCATGTGT-5'

[0054] (2) Recovering the amplified product of step (1) as a template, the following primers were used to truncate the sequence of the CsmiR390a promoter, and cloned products of different truncation forms of the CsmiR390a promoter, P1, P2, P3, and P4, were obtained respectively:

[0055] ProCsmiR390a(P1)-0800-F(SEQ ID NO:13):

[0056] 5'-CGAATTCCTGCAGCCCGGGGGAGTGTGAGGTAACGTAGTCGG-3'

[0057] ProCsmiR390a-0800-R (SEQ ID NO:14):

[0058] 3'-GCTCTAGAACTAGTGGATCGTTTGTATATGTGATGCA-5'

[0059] P2-0800-F (SEQ ID NO: 15):

[0060] 5'-CGAATTCCTGCAGCCCGGGGTCCACAGCTTTACCAAACAAG-3'

[0061] P3-0800-F (SEQ ID NO: 16):

[0062] 5'-CGAATTCCTGCAGCCCGGGGTGCATCACATATACAAACTCA-3'

[0063] P4-0800-F (SEQ ID NO: 17):

[0064] 5'-CGAATTCCTGCAGCCCGGGGTCCACAGCTTTACCAAACAAG-3'

[0065] P4-F (SEQ ID NO:18): 5'-ATTATATATTATTCCACAGCTTTAC-3'

[0066] P4-R (SEQ ID NO:19): 3'-GTAAAGCTGTGGAATAATATATAAT-5'

[0067] (3) Take the pGreenII 0800 vector and perform single enzyme digestion with the restriction endonuclease BamhI to recover the vector backbone.

[0068] (4) The amplified products recovered in steps (1) and (2) and the vector backbone recovered in step (3) were connected to obtain recombinant plasmids ProCsmiR390a::LUC, P1-LUC, P2-LUC, P3-LUC and P4-LUC.

[0069] 3. Transformation of Agrobacterium Competent Cells

[0070] Mix 20 μL of competent cells Agrobacterium GV3101 with 2 uL of plasmid, place on ice for 5 minutes; freeze in liquid nitrogen for 5 minutes; place in a 37°C water bath for 5 minutes; place on ice for 5 minutes, add 500 μL of LB liquid medium (without antibiotics), and culture at 28°C, 220g for 3 hours; draw 200 μL and apply it to a plate containing LB solid medium containing Kan and Rif antibiotics, and culture at 28°C for 3 days; screen positive clones by colony PCR.

[0071] 4. Dual luciferase reporter assay

[0072] (1) Recombinant Agrobacterium containing effectors (35S::Cslnc924 and 35S::Cslnc924tu) and reporter genes (ProCsmiR390a::LUC, P1-LUC, P2-LUC, P3-LUC and P4-LUC) and control recombinant Agrobacterium (pGreenII 0800 vector introduced into GV3101) were activated and cultured to OD600 = 0.8. 600 is 0.8.

[0073] (2) The cells were collected by centrifugation at room temperature and resuspended twice in a solution containing 10 mM MgCl2, 10 mM MES, and 100 μM acetosyringone (pH 5.6);

[0074] (3) Adjust the OD of Agrobacterium suspension 600 The pH was adjusted to 0.8 and used to infiltrate 5-6 week old Nicotiana benthamiana leaves. The plants were then incubated for another 3 days.

[0075] (4) Luciferase substrate was sprayed onto Nicotiana benthamiana leaves, and the luminescence signal was detected using a CCD imaging device (Lumazone Pylon 2048B). Simultaneously, the activities of firefly LUC and Renilla (REN) were measured using a dual-luciferase assay reagent (Promega, Madison, WI, USA). The LUC / REN ratio represents the promoter activity.

[0076] Secondly, based on Agrobacterium-mediated transient overexpression technology and antisense oligonucleotide-mediated gene silencing technology, the Cslnc924 gene was overexpressed and silenced in tea leaves, respectively, and anthracnose inoculation experiments were carried out:

[0077] The specific steps include:

[0078] 1. Transient overexpression in tea leaves

[0079] (1) Activate and culture the recombinant Agrobacterium of 35S::Cslnc924 and 35S::Cslnc924tu to OD 600 is 0.8;

[0080] (2) The cells were collected by centrifugation at room temperature and resuspended twice in a solution containing 10 mM MgCl2, 10 mM MES, and 100 μM acetosyringone (pH = 5.6);

[0081] (3) Adjust the OD of Agrobacterium suspension 600 To 0.8 injection of 108 tea leaves.

[0082] (4) 24 hours later, anthrax inoculation and gene quantitative detection and analysis

[0083] 2. Antisense oligonucleotide silencing experiment of Cslnc924

[0084] (1) SOLIGO software was used to select and synthesize the following candidate antisense oligonucleotides (AsODNs) targeting Cslnc924 by General Biosystems.

[0085] Cslnc924-sODN (SEQ ID NO:20): 5'-GGGCTGCCCAAGGGGGTGACCAAGC-3';

[0086] Cslnc924-AsODN1 (SEQ ID NO:21): 3'-GTCGGACCGTGGTAGCAGGACAG-5';

[0087] Cslnc924-AsODN2 (SEQ ID NO:22): 3'-CCATTTGTCCGATGCATATT-5';

[0088] Cslnc924-AsODN3 (SEQ ID NO:23): 3'-GCTTGGTCACCCCCTTGGGCAGCCC-5'.

[0089] (2) 1 mL of a 100 μM AsODN mixed solution was injected into the leaves of 2-year-old cutting seedlings, while those injected with sense oligonucleotides (sODNs) served as controls;

[0090] (3) Real-time fluorescence quantitative PCR (qRT-PCR) detection and analysis were performed 48 hours later.

[0091] 3. Anthracnose infection of Zhongcha 108 tea leaves

[0092] (1) Cultivate anthrax strains (Camelliae camelliae) on PDA medium at 28°C for 5 days;

[0093] (2) Collect spores by centrifugation at 6000 g for 10 minutes. After collection, resuspend the spores in sterile water and adjust the concentration to 10^6 spores / mL under microscopic monitoring for subsequent inoculation.

[0094] (3) A total of 50 μL of conidia suspension was inoculated into the upper epidermis of tea leaves using a sterile syringe. Control plants were inoculated with an equal amount of sterile distilled water. The inoculated leaves were covered with plastic film to maintain high humidity and promote fungal growth.

[0095] (4) After a 24-hour incubation period, the films were removed and the symptoms of infected leaves were recorded within 6 days after inoculation.

[0096] 3. qRT-PCR assay to detect CsmiR390a, Cslnc924 and CsARFs gene expression levels

[0097] (1) Based on the gene sequence and mature body sequence of the local transcriptome CsmiR390a, the following reverse transcription primers and fluorescence quantitative primers were set using the stem-loop method, with the CsU6 gene as the internal reference.

[0098] CsmiR390a-cDNA (SEQ ID NO:24):

[0099] 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGGCGCT-3'

[0100] CsmiR390a-qF (SEQ ID NO:25): 5'-GCGGCAAGCTCAGGAGGGAT-3';

[0101] CsmiR390a-qR (SEQ ID NO:26): 3'-CCAGTGCAGGGTCCGAGGTA-3';

[0102] CsU6-qF (SEQ ID NO:27): 5'-CGGGGACATCCGATAAAATTG-3';

[0103] CsU6-qR (SEQ ID NO:28): 3'-GGACCATTTCTCGATTTGTGC-3';

[0104] (2) qRT-PCR primers for Cslnc924 and CsARFs were designed according to the MIQE guidelines as follows:

[0105] Cslnc924-qF (SEQ ID NO:29): 5'-ATCTTCTTGTGGTAGGTTGCT-3';

[0106] Cslnc924-qR (SEQ ID NO:30): 3'-TTGGGTTGTTTGAATGTTAAA-3';

[0107] CsARF2.1-qF (SEQ ID NO:31): 5'-GAGATAGGAGCCGATAAAGTTGG-3';

[0108] CsARF2.1-qR (SEQ ID NO:32): 3'-GCAATGAAAGTGCCCTTGAAAGC-3';

[0109] CsARF2.2-qF (SEQ ID NO:33): 5'-CAATGCCTTCTGGTCTCTCCC-3';

[0110] CsARF2.2-qR (SEQ ID NO:34): 3'-TTTCGTTGCTGTGCTCTGCT-3';

[0111] CsARF3-qF (SEQ ID NO:35): 5'-ATCTGCTGACTACTGGTTGGA-3';

[0112] CsARF3-qR (SEQ ID NO:36): 3'-AAGGCTCTTTGAGAATTTACG-3';

[0113] CsARF4.1-qF (SEQ ID NO:37): 5'-AAGAAGAATGCATGTGGCAAT-3';

[0114] CsARF4.1-qR (SEQ ID NO:38): 3'-GGTGGAAAAGGTGAGGAAGAG-3';

[0115] CsGAPDH-qF (SEQ ID NO:39): 5'-TTGGCATCGTTGAGGGTCT-3';

[0116] CsGAPDH-qR (SEQ ID NO:40): 3'-CAGTGGGAACACGGAAAGC-5'.

[0117] (3) The qRT-PCR reaction mixture includes 1 μL cDNA template, 10 μL MonAmp TM ChemoHS qPCR Mix and 1 μL of gene-specific primers were added, and RNA-free water was added to a final volume of 20 μL. The reaction program was as follows: 95°C pre-denaturation for 10 min, 95°C denaturation for 10 sec, 60°C annealing for 10 sec, and 72°C extension for 30 sec, for 40 cycles. The instrument default setting was used for melting curve acquisition. Three biological replicates were performed for each material, and three technical replicates were performed for each PCR reaction. Relative gene expression was calculated using the Pfaffl method.

[0118] The results showed that when Cslnc924 was overexpressed, the Cslnc924 and CsmiR390a genes were significantly upregulated compared with the control, while their downstream CsARFs (CsARF2.1, CsARF2.2, CsARF3 and CsARF4.1) were significantly downregulated. At this time, the resistance of tea plants to anthracnose was enhanced, and the lesion area was significantly reduced (such as Figure 2 In contrast, when Cslnc924 was silenced, the Cslnc924 and CsmiR390a genes were significantly downregulated, while the transcription levels of their downstream CsARFs (CsARF2.1, CsARF2.2, CsARF3, and CsARF4.1) were significantly upregulated. At this time, the resistance of tea plants to anthracnose was weakened, and the lesion area was significantly increased (as shown in Figure 2). Figure 2Notably, when Cslnc924tu, which lacks the CsmiR390Pro binding site, was overexpressed in tea plants, the expression levels of CsmiR390a and its downstream CsARFs (CsARF2.1, CsARF2.2, CsARF3, and CsARF4.1) remained unchanged, and lesion size was not significantly different from the control. These results further confirm that the 257bp-511bp region in Cslnc924 plays a key role in its resistance-conferring function.

[0119] Overall, this study identified a long noncoding RNA (lncRNA) called Cslnc924 that regulates tea plant resistance to anthracnose. Specifically, Cslnc924, a lncRNA significantly upregulated by anthracnose, activates the transcription of CsmiR390a, positively regulating the miR390-TAS3-ARF2s module, thereby reducing CsARF2s expression and enhancing tea plant resistance to anthracnose.

[0120] SEQ ID NO.1:

[0121]

[0122] SEQ ID NO.2:

[0123]

[0124] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A use of the Cslnc924 gene, characterized in that: The expression of the Cslnc924 gene is promoted to enhance the resistance of tea plants to anthrax. The full-length cDNA sequence of the Cslnc924 gene is shown in SEQ ID No.

1.

2. The use of the Cslnc924 gene according to claim 1, characterized in that The Cslnc924 gene is a long non-coding RNA, and its RNA sequence is shown in SEQ ID No.

2.

3. Use of a reagent for detecting the expression level of the Cslnc924 gene according to claim 1 in diagnosing the strength of tea tree anthracnose resistance.

4. Use of the Cslnc924 gene according to claim 1 in the preparation of a medicament for preventing and treating tea tree anthracnose.

5. Use of the Cslnc924 gene according to claim 1 in cultivating anthracnose-resistant tea varieties.

6. A drug for preventing and treating tea tree anthracnose, characterized in that: Comprising a reagent for promoting the expression of the Cslnc924 gene according to claim 1.

7. A tea tree model sensitive to anthrax, characterized in that The anthrax-sensitive tea tree model contains a product that inhibits the expression of the Cslnc924 gene according to claim 1.

8. An expression vector, characterized in that It contains the Cslnc924 gene according to claim 1.

9. A method for breeding anthracnose-resistant tea trees, characterized in that: The following steps are involved: By promoting the expression of the Cslnc924 gene in the target plant, a tea plant with higher resistance to anthracnose than the target plant is obtained.

10. The method for breeding anthracnose-resistant tea trees according to claim 9, characterized in that: The method for promoting the expression of the Cslnc924 gene in the target plant comprises: constructing a Cslnc924 gene overexpression vector and introducing the vector into the target plant.

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